The nucleotide excision repair pathway (NER) is a highly versatile DNA repair pathway present in a number of organisms from bacteria to mammals which requires the contribution of over thirty proteins. The NER pathway repairs a wide array of bulky DNA damage from a variety of sources such as, reactive chemicals and exposure to UV light. Numerous non-enzymatic protein-DNA interactions are essential for the proper functioning of the NER machinery and play important roles in nearly every reaction in the pathway including lesion recognition. Damaged DNA is recognized by the trimeric complex consisting of Xeroderma Pigmentosum Group C (XPC), Rad23B and Centrin 2 during global genomic nucleotide excision repair (GG-NER) while the stalling of RNA polymerase during transcription is the method of damage recognition during transcription-coupled (TC) NER. Following damage recognition the preincision NER complex is completed with the subsequent recruitment of Xeroderma Pigmentosum Group A (XPA) protein, Transcription Factor II H (TFIIH) protein and the human single-stranded DNA (ssDNA) binding protein, Replication protein A (RPA) to the site of DNA damage. RPA is one of the first proteins that functions in both the GG and TC-NER subpathways. RPA is a heterotrimeric DNA binding protein containing three subunits p70, p34, and p14 (kDa) and plays an important role in DNA replication and recombination in addition to repair. The p70 RPA subunit contains DNA binding domains A and B (DBD-A and DBD-B) and contributes most significantly to the RPA-ssDNA interaction. The RPA p34 subunit also contains an OB-fold and interacts with additional proteins including XPA while the 14 kDA subunit plays a role in protein stability. The RPA-DNA interaction is essential for the formation of the NER preincision complex and proper functioning of the NER pathway. Disruption of this essential protein-DNA interaction via small molecule inhibitors (SMIs) should reduce the NER efficiency. Previous reports have demonstrated that decreased expression levels of essential NER proteins, such as XPA result in decreased NER capacity and removal of cisplatin adducts. Furthermore, increased expression of ERCC1-XPF was demonstrated to correlate with cisplatin resistance in ovarian cancer cell lines. Taken together, these data suggest that expression level of essential NER proteins affects the efficiency of the NER machinery. Using SMIs to inhibit RPA-DNA interactions and consequently the function of the NER machinery may increase the efficacy of DNA-damaging chemotherapeutics, particularly in tissues where enhanced repair via NER is a resistance mechanism.
The importance of RPA in DNA replication has been demonstrated by genetic studies in yeast, genetic knockdown studies in human cells and more recently in chemical genomic studies with a small molecule inhibitor of RPA. RPA plays multiple roles in DNA replication including assembly of pre-replication complexes and stabilization of ssDNA following helicase-catalyzed unwinding. Moreover, very recent data demonstrating that RPA can unwind duplex DNA has led to a model where RPA may help in maintaining double stranded DNA stability throughout replication. Inhibition of any one of these steps is likely to have deleterious effects on DNA replication and ultimately cell viability.
RPA inhibition with a recently identified SMI of RPA, TDRL-505, has been demonstrated to synergize with cisplatin in a human lung cancer cell model (1). This effect is likely to be a function of alterations in DNA repair, specifically nucleotide excision repair (NER), though effects on homologous recombination cannot be ruled out. Cisplatin, cis-diamminedichloroplatinum (II), is commonly used as a chemotherapeutic drug in cancer treatment that forms cytotoxic intra- and inter-strand DNA-cisplatin adducts. DNA-cisplatin adducts are repaired mainly through the NER pathway and RPA has been shown to preferentially bind to duplex cisplatin-damaged DNA compared to undamaged DNA through the development of ssDNA. RPA is also responsible for the recognition of inter-strand cross-links caused by cisplatin treatment. Cisplatin resistant cancers have been linked to enhanced DNA repair and thus the ability to impact DNA repair efficiency via modulation of RPA's DNA binding activity is of potential clinical use to treat cancer in conjunction with platinum agents. Etoposide, a common chemotherapeutic drug that induces replication fork stalling by inhibiting topoisomerase II, was also demonstrated to synergize with the RPA SMI TDRL-505 (1). This synergistic activity is predicted to increase the toxic effects exerted by etoposide both in the context of replication and DNA repair. RPA's role in homologous recombination may be mediating this effect where DNA double strand breaks are processed to generate a 3′ ssDNA overhang to which RPA binds to help catalyze RAD51 dependent strand exchange. In Saccharomyces cerevisiae, mutations within the DNA binding domain and protein-protein interaction regions of ScRPA lead to highly decreased meiotic recombination. This is consistent with data obtained from SMIs of hRPA demonstrating decreased DNA replication, repair and recombination in cancer cells and increased efficacy of treatments with DNA damaging agents. Given, RPA's role in cancer cell drug resistance there is a need for materials and methods for regulating this enzymes activity in some cells, some embodiments of the invention disclosed herein address this need.